Virulence of streptococci

ABSTRACT

The invention relates to the field of diagnosis of and vaccination against Streptococcal infections and to the detection of virulence markers of Streptococci. The invention discloses a method for modulating virulence of a  Streptococcus , the method comprising modifying a genomic fragment of  Streptococcus  wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in  Streptococcus suis  to a nucleic acid or fragment thereof as shown in FIG.  5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/435,654, filed May 9, 2003, now U.S. Pat. No. 7,109,006, whichapplication is a continuation of PCT/NL01/00805, filed Nov. 6, 2001,designating the United States of America, corresponding to PCTInternational Publication WO 02/38597 (published in English on May 16,2002), the contents of each of which are incorporated herein in itsentirety.

TECHNICAL FIELD

The invention relates generally to biotechnology, and, moreparticularly, to the diagnosis of and vaccination against Streptococcalinfections and to the detection of virulence markers of Streptococci.

BACKGROUND

Streptococcus species, of which there are a large variety of that causeinfections in domestic animals and man, are often grouped according toLancefield's groups. Typing, according to Lancefield, occurs on thebasis of serological determinants or antigens that are, among others,present in the capsule of the bacterium and, thus, allows for anapproximate determination. Often bacteria from a different group showcross-reactivity with each other, while other Streptococci cannot beassigned a specific group-determinant at all. Within groups, furtherdifferentiation is often possible on the basis of serotyping. Theseserotypes further contribute to the large antigenic variability ofStreptococci, a fact that creates an array of difficulties withindiagnosis of and vaccination against Streptococcal infections.

Lancefield group A Streptococcus (GAS, Streptococcus pyogenes), arecommon with children and cause nasopharyngeal infections andcomplications thereof. Among animals, cattle are susceptible to GAS, andmastitis is often found.

Lancefield group B Streptococcus (GBS) are most often seen with cattleand cause mastitis. However, human infants are susceptible as well,often with fatal consequences. Group B streptococci (GBS) constitute amajor cause of bacterial sepsis and meningitis among human neonates bornin the United States and Western Europe and are emerging as significantneonatal pathogens in developing countries.

Lancefield group C infections, such as those with S. equi, S.zooepidemicus, S. dysgalactiae, and others are mainly seen with horse,cattle and pigs, but can also cross the species barrier to humans.

Lancefield group D (S. bovis) infections are found with all mammals andsome birds, sometimes resulting in endocarditis or septicemia.

Lancefield groups E, G, L, P, U and V (S. porcinus, S. canis, S.dysgalactiae) are found with various hosts, cause neonatal infections,nasopharyngeal infections or mastitis.

Within Lancefield groups R, S, and T (and with ungrouped types), S. suisis found and is an important cause of meningitis, septicemia, arthritisand sudden death in young pigs. Incidentally, it can also causemeningitis in man.

Ungrouped Streptococcus species, such as S. mutans, causes caries withhumans, S. uberis, causes mastitis with cattle, and S. pneumonia, causesmajor infections in humans, and Enterococcus faecalis and E. faecium,further contribute to the large group of Streptococci. Streptococcuspneumoniae (the pneumococcus) is a human pathogen that causes invasivediseases, such as pneumonia, bacteremia, and meningitis.

Little is known about the pathogenesis of the disease caused byStreptococci. Various cellular components, such as muramidase-releasedprotein (MRP), extracellular factor (EF) and cell membrane associatedproteins, fimbriae, hemagglutinins, and hemolysin have been suggested asvirulence factors. However, the precise role of these protein componentsin the pathogenesis of the disease remains unclear. It is, however,known and generally accepted that the polysaccharidic capsule of variousStreptococci and other gram-positive bacteria plays an important role inpathogenesis. The capsule enables these microorganisms to resistphagocytosis and is, therefore, regarded as an important virulencefactor or marker.

In particular, Streptococcus suis is an important cause of meningitis,septicemia, arthritis and sudden death in young pigs. It can also causemeningitis in man. Attempts to control the disease are hampered by thelack of sufficient knowledge about the pathogenesis of the disease andthe lack of effective vaccines and sensitive diagnostic methods.

So far, 35 serotypes of S. suis have been described. Virulence of S.suis can differ within and among serotypes. Worldwide, S. suis serotype2 is the most frequently isolated serotype. Within S. suis serotype 2,pathogenic, weak-pathogenic and non-pathogenic strains can be found. Thepathogenic strains cause severe clinical signs of disease in pigs andlarge numbers of bacteria can be re-isolated from the central nervoussystem (CNS) and the joints after experimental infection. Theweak-pathogenic strains cause only mild clinical signs of disease andinfrequently bacteria can be re-isolated from the CNS and the jointsafter experimental infection. The non-pathogenic strains are completelyavirulent in young pigs after experimental infection.

The 136-kDa muramidase-related protein (MRP) and the 110-kDaextracellular factor (EF) are generally considered as importantvirulence markers for S. suis serotype 2 strains isolated in Europe andthe United States. However, differences in virulence between pathogenic,weak-pathogenic and non-pathogenic strains cannot exclusively beexplained by differences in their MRP and EF expression patterns. Inaddition, it is known that the capsule of Streptococcus suis serotype 2is an important virulence factor. However, since pathogenic,weak-pathogenic and non-pathogenic strains seem to be fully encapsulatedafter growth in vitro and in vivo, it is not likely that the level ofencapsulation of these fully encapsulated strains is associated withtheir difference in virulence.

SUMMARY OF THE INVENTION

Disclosed are methods for modulating virulence of a Streptococcuscomprising modifying a genomic fragment of the Streptococcus, whereinthe genomic fragment comprises at least a functional part of a fragmentidentifiable by hybridization in Streptococcus suis to a nucleic acid orfragment thereof as shown in FIG. 5. To gather an insight into thedifferences between pathogenic, weak-pathogenic and non-pathogenicstrains that determine their difference in virulence, the inventiondiscloses an in vivo complementation system wherein virulence can bemodified by modifying the fragment.

For example, within S. suis serotype 2, pathogenic, weak-pathogenic andnon-pathogenic strains are found. A genomic library of a pathogenicstrain was introduced into a weak-pathogenic strain. After infection ofthe library into young piglets, pathogenic transformants were selected.One specific transformant that contained a 3 kb fragment of thepathogenic strain, V10, appeared to be dominantly enriched in diseasedpigs. The observed enrichment was not tissue specific. The selectedfragment, when introduced into two different weak-pathogenic strains,considerably increased the virulence of these strains. In particular,the fragment described and identified as ORF2, or functional fragmentsthereof, was shown to be an important virulence factor. In contrast,introduction of the corresponding fragment of a weak-pathogenic strainhad only minor effects on virulence.

Accordingly, also described are methods for assaying virulence of aStreptococcus comprising assaying a genomic fragment of theStreptococcus, wherein the genomic fragment comprises at least afunctional part of a fragment identifiable by hybridization inStreptococcus suis to a nucleic acid or fragment thereof as shown inFIG. 5, in particular the ORF2 fragment.

Nucleotide sequence analysis of the selected fragment of the pathogenicstrain revealed the presence of two potential open reading frames, bothof which were found to be mutated in the corresponding fragment of theweak-pathogenic strain. It was previously shown by ribotyping and randomamplified polymorphic DNA analysis (RAPD) assays that pathogenic andweak-pathogenic strains of S. suis serotype 2 are genetically closelyrelated, whereas non-pathogenic strains showed a high degree of geneticheterogeneity. A genomic library of the pathogenic S. suis strain 10 inplasmids was constructed and the plasmid library was introduced into theweak-pathogenic reference strain of S. suis serotype 2, strain S735.Pigs were inoculated intravenously with the recombinants and bacteriawere recovered from the CNS and the joints of diseased pigs.

The re-isolated bacteria were subsequently analyzed for plasmid contentand virulence. With this approach, a DNA fragment of a pathogenicserotype 2 strain that transformed weak-pathogenic strains into highlypathogenic strains was identified. This fragment, as described herein,comprises a genetic determinant important for virulence. The fragment isin other Streptococci identifiable by, for example, hybridizationexperiments such as Northern or Southern blotting, or by amplificationexperiments (such as PCR) using primers and/or probes derived from anucleic acid as described herein.

With the fragment and parts thereof, such as the open reading framesidentified in FIG. 3, a virulence marker is described herein. The markeris associated with an isolated and/or recombinant nucleic acid asdescribed herein and derivable from Streptococcus and identifiable byhybridization in Streptococcus (preferably S. suis) to a nucleic acid orfragment thereof as shown in FIG. 5.

Also described are vectors comprising a nucleic acid according to theinvention and a host cell comprising a nucleic acid or a vectoraccording to the invention. Such a host cell comprises an easilymodifiable organism such as E. coli. However, other host cells, such asrecombinant Streptococcus (such as those derived from one of the groupedor ungrouped Streptococci as identified hereinabove) comprising a vectoror nucleic acid according to the invention are also herein disclosed. Inparticular, recombinant Streptococcus as described herein is useful forinclusion in a vaccine.

Furthermore, also described are vaccines comprising a nucleic acid, avector or a host cell according to the invention, and the use of such avaccine in the prevention and/or treatment of Streptococcal infections.

Also described is a protein or fragment thereof encoded by a nucleicacid according to the invention, such as a protein encoded by ORF2 orORF3 as disclosed herein, or a functional, i.e., antigenic fragmentthereof. The invention also discloses an antibody directed against aprotein or fragment thereof according to the invention and an antigenreactive with such an antibody, for example, comprising a protein orfragment. Such a protein or fragment thereof need not be obtained byrecombinant means since synthesis of the peptides according to the aminoacid sequence is also possible. Such antigens and antibodies asdescribed herein can be used in a diagnostic test comprising an antibodyof the invention, or within a vaccine or diagnostic test comprising theantigen of the invention. Such vaccines and diagnostic tests can be usedin the field of diagnosis of and vaccination against Streptococcalinfections and for the detection of virulence markers of Streptococci.

The phrase “means for imparting virulence” will be used to refer to anucleic acid that encodes a peptide corresponding to a virulence factoror to a peptide encoded by the nucleic acid that possesses acharacteristic associated with the virulence factor. The phrase “meansfor imparting virulence” also includes, without limitation, the nucleicacids having sequences corresponding to SEQ ID NO:14 and SEQ ID NO:15,and the peptides having sequences corresponding to SEQ ID NOS:10-13, andany functional sequences originating therefrom. For instance, thenucleic acid sequence and the amino acid sequence may have conservativechanges, such as additions, deletions or substitutions that do notaffect a function associated with the virulence factor. For example,since the genetic code is degenerate, i.e., an amino acid may be encodedfor by more than one codon, a conservative change in an original nucleicacid may result in an altered nucleic acid that encodes the same orhomologous peptide as the original nucleic acid, wherein the peptideencoded by the altered nucleic acid retains the same function as theoriginal nucleic acid. Further, since some amino acids are similar incharge, a conservative change, such as an addition, deletion orsubstitution, in the original amino acid sequence may result in analtered amino acid sequence, wherein the altered amino acid sequenceretains the same function as the original amino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the pCOM1 vector. pCOM1 contains the replicationfunctions of pWVO1, the erythromycin-resistance gene of pE194 precededby the promoter region of the mrp gene, as well as the SacI-PstI part ofthe multiple cloning site of pKUN19.

FIG. 2 shows plasmids digested with SmaI and XbaI on an 0.8% agarosegel. Library: plasmids isolated from ten randomly selected clones of theoriginal library; clones enriched in pigs: plasmids isolated from 19independently selected clones enriched in pigs; c: pCOM1; m: molecularsize marker.

FIG. 3 is a schematic representation of the fragments V10 and V735. Thearrows indicate the potential ORFs. æ P, indicates the position of thepotential promoter sequence; °, indicates the positions of the potentialtranscription regulator sequences. Homologies (% identities) between thepotential proteins encoded by the ORFs and proteins present in the datalibraries are indicated.

FIGS. 4A and 4B illustrate the homology between the ORFs 2 (A) and 3 (B)encoding proteins of fragments V10 and V735. The asterisks indicate thenon-identical amino acids.

FIGS. 5A-5F are nucleotide and amino acid sequences of fragments V10 andV735.

DETAILED DESCRIPTION

Bacterial strains and growth conditions. The bacterial strains andplasmids used herein are listed in Table 1. S suis strains were grown inTodd-Hewitt broth (code CM189, Oxoid), and plated on Columbia agar bloodbase (code CM331, Oxoid) containing 6% (v/v) horse blood. If required,antibiotics were added at the following concentrations: erythromycin, 1μg/ml. E coli strains were grown in Luria broth and plated on Luriabroth containing 1.5% (w/v) agar. If required, 200 μg/ml of erythromycinwas added.

pCOM1. pCOM1 (FIG. 1) is based on the replication functions of pWVO1.Further, the vector contained the erythromycin-resistance gene of pE194preceded by the promoter region of the mrp gene, as well as theSacI-PstI part of the multiple cloning site of pKUN19. As the result,pCOM1 contained a unique BamHI site (FIG. 1).

Construction of genomic S. suis library in pCOM1. Sau3AI partial digestsof the DNA of the pathogenic S. suis serotype 2, strain 10 were sizefractionated (>3 kb) by precipitation with 4.6% of PEG 6000 (BDHChemicals, 19). The fragments were ligated to BamHI digested pCOM1 andthe ligation mixtures were transformed to E. coli XL2-blue cells.Erythromycin-resistant colonies were selected. About 17,000 independentE. coli clones were obtained. Analysis of 55 of the transformants showedthat 64% contained an insert of greater than 3 kb. From the pool of E.coli transformants, plasmid DNA was isolated and subsequently used forthe electrotransformation of the weak-pathogenic S. suis strain S735.This resulted in approximately 30,000 independent S. suis transformants.The S. suis library was designated S735 (pCOM-L). The transformants werepooled and stored at −80° C.

DNA techniques. Routine DNA manipulations were performed as described bySambrook et al. DNA sequences were determined on a 373A DNA SequencingSystem (Applied Biosystems, Warrington, GB). Samples were prepared byuse of an ABI/PRISM dye terminator cycle sequencing-ready reaction kit(Applied Biosystems). Custom-made sequencing primers were purchased fromLife Technologies. Sequencing data was assembled and analyzed using theMcMolly/Tetra software package. The BLAST program was used to search forprotein sequences homologous to the deduced amino acid sequences.

For PCR reaction mixtures (50 μl), the PCR Expand High Fidelity system(Boehringer, Mannheim, Germany) was used as described by the supplier.DNA amplification was carried out in a Perkin Elmer 9600 thermal cyclerand the program included an incubation for two minutes at 95° C., tencycles of 20 seconds at 95° C., one minute at 60° C. and four minutes at68° C., 30 cycles of 20 seconds at 95° C., one minute at 60° C. and fourminutes, extended with 20 seconds for each cycle, at 68° C. and tenminutes at 72° C.

Southern blotting and hybridization. Chromosomal DNA was isolated asdescribed by Sambrook et al. DNA fragments were separated on 0.8%agarose gels and transferred to Gene-Screen Plus membranes (NEN) asdescribed by Sambrook et al. DNA probes were labeled with [(α-³²P]dCTP(3000 Ci mmol⁻¹; Amersham) by use of a random primed labeling kit(Boehringer). The DNA on the blots was hybridized at 65° C. with theappropriate DNA probes as recommended by the supplier of the Gene-ScreenPlus membranes. After hybridization, the membranes were washed twicewith a solution of 40 mM sodium phosphate, pH 7.2, 1 mM EDTA, 5% SDS for30 minutes at 65° C., and twice with a solution of 40 mM sodiumphosphate, pH 7.2, 1 mM EDTA, 1% SDS for 30 minutes at 65° C.

Construction of pCOM-V10-ORF2 and pCOM-V10-ORF3. To constructpCOM-V10-ORF2, the primers 5′-CGAGCTCGGAAGAATTGGTTATTGCGCGTG-3′ (SEQ IDNO:1) and 5′-CGGGATCCCGGGGGATGACCTGTTGCTTG-3′ (SEQ ID NO:2) were used ina PCR reaction on chromosomal DNA of S. suis strain 10 to amplify theORF2 encoding region. The resulting fragment was purified, digested withSacI and BamHI and cloned into SacI and BamHI-digested pCOM1.

To construct pCOM-V10-ORF3, the primers5′-TCCCCCGGGGGACAAGCAACGGGTCATCCCC-3′ (SEQ ID NO:3) and5′-CGGGATCCCGGTTGAATGCCCGGCAAAGCG-3′ (SEQ ID NO:4) were used to amplifythe ORF3 encoding region. The resulting fragment was digested with SmaIand BamHI and cloned into pKUN19. The resulting plasmid was designatedpKUN-ORF3. Because the ORF2 and ORF3 encoding regions are most probablyco-transcribed, the promoter region of ORF2 was subsequently amplifiedwith primers 5′-CGAGCTCGGAAGAATTGGTTATFGCGCGTG-3′ (SEQ ID NO:1) and5′-TCCCCCGGGGGAGTCGTGTGTATTCGACAGCGG-3′ (SEQ ID NO:5). The fragmentswere digested with SacI and SmaI and cloned into SacI and SmaI digestedpKUN-ORF3. The resulting plasmid was digested with SacI and BamHI, theinsert fragment was purified and cloned into SacI and BamHI digestedpCOM1. This resulted in pCOM-V10-ORF3.

Experimental infections. Germ free pigs, crossbreeds of Great Yorkshireand Dutch landrace, were obtained from sows by caesarian sections. Thesurgery was performed in sterile flexible film isolators. Pigs wereallotted to groups, each including 4 or 5 pigs, and were housed insterile stainless steel incubators. Housing conditions and feedingregimes were performed as described by Vecht et al. One week old pigswere intravenously inoculated with S. suis strains as described by Vechtet al. Pigs received erythromycin orally twice a day (Erythromycinstearate, Abbott B. V., Amstelveen, The Netherlands, 40 mg/kg bodyweight). Two hours after the infection, the pigs were treated witherythromycin for the first time. Pigs were monitored twice a day forclinical signs of disease, such as fever, nervous signs and lameness.Blood samples were collected three times a week from each pig. Whiteblood cells were counted with a cell counter.

To monitor infection with S. suis, swabs of nasopharynx and feces werecollected daily. The swabs were directly plated onto Columbia agarcontaining 6% horse blood. After the pigs were sacrificed, they wereexamined for pathological changes. Further, tissue specimens werecollected from the central nervous system, serosa, joints, lungs, liver,kidney, spleen, heart and tonsils. The tissues were homogenized in thepresence of Todd-Hewitt medium by using an Ultra-Turrax tissuemizer(Omni International, Waterbury, USA), centrifuged for five minutes at3,000 rpm and the supernatants were frozen at −80° C. in the presence of15% glycerol.

Results.

Complementation system. A genomic library of the pathogenic S. suisstrain 10 was constructed into the weak-pathogenic strain S735 asdescribed in Materials and Methods. The plasmid pCOM1 allowed theinsertion of large DNA fragments into the unique BamHI site (FIG. 1).The plasmid carries the origin of replication of pWVO1 that functions inE. coli and in S. suis. This allowed the construction of a DNA libraryin E. coli first. Plasmid DNA, isolated from the pool of E. colitransformants, was subsequently electrotransformed into S. suis strainS735. 30,000 individual S. suis clones were obtained. As determined byanalysis of 24 randomly selected transformants, more than 30% of theS735 (pCOM-L) transformants contained an insert greater than 3 kb.

Selection of genomic fragments associated with virulence. To select forgenetic determinants of the pathogenic S. suis strain 10 that couldincrease the virulence of the weak-pathogenic strain S735, pigs wereinoculated with the S. suis library S735 (pCOM-L). A dose of either 10⁷or 10⁸ cfu was used and the pigs were treated with erythromycin asdescribed in Materials and Methods. All pigs showed specific S. suissymptoms (Table 2, A) three to seven days after the infection and exceptfor one, all pigs died during the course of the experiment. From five ofthe pigs, bacteria could be re-isolated from the CNS and from two otherpigs, bacteria were isolated from the joints (Table 2, A).

In previously performed experiments in which pigs were inoculated withweak-pathogenic strains, specific S. suis symptoms were observed with avery low frequency. In addition, from those pigs, bacteria could not bere-isolated from the CNS or from the joints. Therefore, the dataindicated that, compared to virulence of strain S735, bacteria isolatedfrom pigs inoculated with the S. suis library S735 (pCOM-L) are morevirulent due to the presence of a DNA fragment of the pathogenic strain10. The plasmid content of 90 randomly selected clones isolated from theCNS or the joints of the seven diseased pigs was analyzed by PCR andrestriction analysis. The results showed that 88 of the 90 clonesanalyzed (19 of which are shown in FIG. 2) contained an insert of about3 kb and had substantially identical restriction patterns. Moreover, theinserts of ten randomly selected clones having substantially identicalrestriction patterns, also showed identical DNA sequences (results notshown). Plasmid DNA of ten randomly selected clones from the originalS735 (pCOM-L) library showed ten different restriction patterns (FIG.2). The data suggest that one specific clone, which was designated S735(pCOM-V10), was greatly enriched in seven different pigs. Further, thisparticular clone was isolated from the CNS and from the joints of thevarious pigs, indicating that the observed enrichment was not tissuespecific.

Virulence-associated properties of the selected fragment V10. To furtheranalyze the virulence properties of strain S735 (pCOM-V10), pigs wereintravenously inoculated with 10⁶ cfu of strain S735 (PCOM1) or strainS735 (pCOM-V10). The results (Table 2, B) show that, compared to thevirulence of strain S735 (pCOM1), the virulence of strain S735(pCOM-V10) was greatly enhanced.

All pigs inoculated with strain S735 (pCOM-V10) showed specific S. suissymptoms and died within one day after infection. In contrast, exceptfor one, none of the pigs inoculated with the control strain S735(pCOM1) showed specific clinical symptoms and these pigs survived untilthe end of the experiment (15 days after infection). The data provedthat introduction of fragment V10 of strain 10 into S735 transformed theweak-pathogenic strain S735 into a highly pathogenic strain. Thisstrongly suggests that the protein(s) encoded by V10 are importantvirulence determinants and play an important role in the pathogenesis ofS. suis serotype 2 infections in pigs.

To find out whether the observed increase of the fragment V10 onvirulence was specific for strain S735, pCOM1 and pCOM-V10 wereintroduced into another weak-pathogenic strain, strain 24. Subsequently,the virulence properties of the strains 24 (pCOM1) and 24 (pCOM-V10)were determined. As shown in Table 2 C and D, similar effects of V10 onthe virulence of strains S735 and 24 were observed. Both strains 24(pCOM-V10) and S735 (pCOM-V10) were highly pathogenic for young piglets,whereas strains 24 (pCOM1) and S735 (pCOM1) were shown to beweakly-pathogenic (Table 2, C and D). This strongly indicates that V10has a more general ability to transform weak-pathogenic serotype 2strains into highly pathogenic strains.

Because a plasmid system for the complementation approach was used,gene-dose effects cannot be excluded. Plasmid pCOM1 is based on thereplication functions of pWVO1. In Gram-positive bacteria, the latterplasmid has a copy number of between 3 and 6. To find out whether copyeffects play a role, the genomic region of strain S735 homologous tofragment V10 of strain 10 (see below) was cloned into plasmid pCOM1.This plasmid was designated pCOM-V735. The virulence of strains S735(pCOM-V735), and 24 (pCOM-V735) was subsequently compared to that ofS735 (pCOM-V10), S735 (pCOM1), 24 (pCOM-V10) and 24 (pCOM1). The results(Table 2, C and D) show that, in contrast to pCOM-V10, the plasmidpCOM-V735, did not carry virulence-enhancing activity. Pigs infectedwith strains S735 (pCOM-V10) and 24 (pCOM-V10) died within one or twodays after infection, whereas most of the pigs infected with strainsS735 (pCOM-V735), 24 (pCOM-V735), S735 (pCOM1) and 24 (pCOM1) surviveduntil the end of the experiment (17 days after infection).

Compared to pigs infected with strains containing pCOM1, pigs infectedwith strains containing pCOM-V735 developed more general and specificsigns of disease, but much less than pigs infected with strainscontaining pCOM-V10 (Table 2, C and D). From these data, it wasconcluded that the differences in virulence observed between the strainscontaining pCOM-V1 and the strains containing pCOM-VS735 are caused bydifferences between the fragments V10 and V735 (see below). Thedifferences in virulence observed between the strains containing pCOM1and the strains containing pCOM-VS735 may be due to gene-dose effects.

Sequence analysis of fragments V10 and V735. By using the fragment V10as a probe, a 3.1 kb PstI-HindIII fragment of strain S735 (V735) wasidentified and cloned into pCOM1 (FIG. 3). To analyze the differencesbetween the fragments V10 and V735, the nucleotide sequences of thefragments V10 and V735 were determined and the sequences were analyzedfor homology to known genes by comparison with the GenBank/EMBL andSWISSPROT databases.

The sequence of V10 revealed two complete and two incomplete openreading frames (FIG. 3). ORF1 (nucleotides 1 to 461) coded for apolypeptide of 153 amino acids. This protein showed homology (49%identity) to the C-terminal region of acetate kinase of Clostridiumthermocellum (accession number AF041841) and various other bacterialspecies. ORF2 (nucleotides 625 to 1327) coded for a protein of 233 aminoacids. No significant similarities were found between the predictedamino acid sequence of this protein and other proteins present in thedata libraries.

ORF3 (nucleotides 1382 to 2639) coded for a protein of 418 amino acids.This protein showed homology (36% identity) to FolC (folylpolyglutamatesynthetase) of Bacillus subtilis. Compared to the other ORFs, ORF4 istranscribed in the opposite direction. ORF4 (nucleotides 2684 to 2972)coded for a polypeptide of 96 amino acids. This polypeptide showedhomology (67% identity) to the C-terminal part of PepA(glutamyl-aminopeptidase) of Lactococcus lactis. Both ORFs 2 and 3possessed putative initiation codons and ribosome-binding sites.Putative −35 (TGGACA) and −10 (TACAAT) sequences, which may function aspromoter sequences, were found preceding ORF2. ORFs 2 and 3 wereseparated by 55 nucleotides. In this region, no putative promotersequences could be observed. This could indicate that the ORFs 2 and 3are co-transcribed. Downstream of the ORFs 1 and 3, regions of extendeddyad symmetry were found which may function as transcription terminationsignals.

The sequence of the fragment V735 was determined and compared to thesequence of the fragment V10. No major deletions or insertions werefound between the sequenced regions. The ORFs 1, 3 and 4 of strains 10and S735 were highly homologous. The putative protein fragments encodedby the ORFs 1 differed in 2 (1.3%) amino acids; the putative proteinsencoded by the ORFs 3 differed in 19 (4.5%) amino acids (FIG. 4B),whereas the putative protein fragments of the ORFs 4 were identical.However, major differences were observed between the ORFs 2 of strains10 and S735. In the pathogenic strain 10, an ORF of 699 bases was foundwith a protein product of 233 amino acids. In contrast, due to aframe-shift mutation in the weak-pathogenic strain S735, an ORF of 569bases was found and coded for a polypeptide of 183 amino acids.

Compared to the putative protein encoded by strain 10, the putativeprotein encoded by strain S735 lacked the N-terminal 50 amino acids(FIG. 4A). Beside these N-terminal differences, the putative proteinsdiffered at 9 amino acid positions (4.9%). In addition, the putative −35regions that may be part of the promoter sequences involved in theexpression of ORFs 2 and 3, differed between the two strains. A TGGACAsequence was found in strain 10, whereas a TGGTCA sequence was found instrain S735. The sequence data suggest that the differences in thevirulence-enhancing effects of the fragments V10 and V735 may be theresult of functional differences between the putative proteins expressedby the ORFs 2 and/or 3, and/or by differences in their levels ofexpression.

ORF2 or ORF3.

To examine whether the observed increase of the fragment V10 onvirulence resulted from ORF2 or ORF3 or both, the plasmids pCOM-V10-ORF2and pCOM-V10 ORF3 containing the individual ORF2 and ORF3 encodingregions were constructed. Because ORF3 is probably co-transcribed withORF2, in pCOM-V10-ORF3 the ORF3 encoding region was preceded by thepromoter region of ORF2. Subsequently, the virulence properties of thestrains S735 (pCOM-V10), S735 (pCOM-V10-ORF2), S735 (pCOM-V10-ORF3) andS735 (pCOM1) were determined. As shown at E in Table 2, the fragmentsV10 and ORF2 showed similar effects on the virulence of strain S735while no effect of ORF 3 could be observed on the virulence of strainS735. These data show that ORF2 is responsible for the observed effecton virulence and that the ORF2 protein is an important virulence factor.

Distribution of the ORF2 and ORF3 sequences among all known 35 S. suisserotypes. To examine the homology between the ORF2 and ORF3 genes andgenes of other S. suis serotypes, cross-hybridization experiments wereperformed. DNA fragments of the ORF2 and 3 genes were amplified by PCR,labeled by ³²P, and hybridized to chromosomal DNAs of the referencestrains of the 35 different S. suis serotypes. As a positive control, aprobe specific for 16S rRNA was used. The 16S rRNA probe hybridized withalmost equal intensities with all serotypes tested (results not shown).Probes ORF2 and ORF3 hybridized with all serotypes, except for serotypes32 and 34 (results not shown). This indicates that the proteins encodedby ORF2 and 3 are common among most Streptococcus species.

Herein, the development and the successful application of an in vivocomplementation approach for the identification of important moleculardeterminants that determine the differences in virulence betweenpathogenic and weak-pathogenic strains of Streptococcus is described.Using the complementation approach, one unique clone containing a 3.0 kbfragment of pathogenic strain (V10) was selected. The selected fragmentwas greatly enriched in seven different pigs and the observed enrichmentwas not tissue specific. The selected fragment showed similar enhancingeffects on the virulence of two different weak-pathogenic strains. Largedifferences were observed between the effects of the selected fragmentV10 of the pathogenic strain 10 and the corresponding fragment V735isolated from the weak-pathogenic strain S735 on virulence.

In contrast to V10 which had a strong virulence-enhancing effect onweak-pathogenic strains, V735 showed only minor effects. Therefore,differences between these two fragments are considered responsible forthe observed differences on virulence. Sequence data showed that thefragments V10 and V735 were highly homologous. Both fragments containedtwo complete ORFs (ORFs 2 and 3), both of which can potentially expressproteins that may further contribute to the observed effect onvirulence. The ORFs 3 are highly homologous and differ in only 19 aminoacids.

The proteins encoded by the ORFs 3 showed homology to FolC(folylpolyglutamate synthetase) of various pro- and eukaryoticorganisms. Folylpolyglutamate synthetase catalyzes the conversion offolates to polyglutamate derivatives. Bacteria require folates for thebiosynthesis of glycin, methionine, formylmethionine, thymidine, purinesand pantothenate. Whether the FolC proteins encoded by the fragments V10and V735 have different enzymatic activities or different substratespecificities is unknown so far. In E. coli, a folC mutant is methioninedeficient, however, so far a role of FolC in virulence has not beendescribed. Significant differences were also observed between the ORFs 2of the fragments V10 and V735. Compared to the putative ORF2 proteinencoded by strain 10, the putative protein encoded by strain S735 lackedthe N-terminal 50 amino acids. In strain S735, a strong ribosome-bindingsite precedes the methionine start codon of ORF2. In contrast, however,the sequence in strain 10 did not indicate the presence of a strongribosome-binding site preceding the methionine start codon of ORF2.Therefore, although ORF2 of strain 10 is extended compared to ORF2 ofstrain S735, it is not clear whether the proteins expressed by these twoORFs differ in length.

In addition to the putative N-terminal differences, the putative ORF2proteins differed at nine amino acid positions (4.9%). Except for oneamino acid, these amino acid substitutions were clustered at twodifferent positions in the putative protein. The function of the ORF2protein is unknown so far. Not even distant or partial homologies werefound between the ORF2 protein sequences and protein sequences presentin the data libraries. Hydrophobicity profiles showed that the ORF2encoded protein(s) are very hydrophobic thus suggesting a role of theORF2 protein in the cellular membrane. The putative-35 region precedingthe ORFs 2 and 3 differed between strains S735 and 10. Therefore,differences in the expression levels rather than functional differencesresponsible for the observed effects on virulence are not excluded.

In previous experiments, it was found that pigs infected withweak-pathogenic strains showed only mild clinical signs of disease andthat bacteria could never be re-isolated from the CNS or the joints.Surprisingly, in the experiments described herein in whichweak-pathogenic strains containing the control plasmid pCOM1 were used,bacteria could (with a low frequency) be re-isolated from the CNS aswell as from the joints. Several possible explanations for theseobserved differences exist. One explanation is that the presence of theplasmid somehow affects the (virulence) properties of the strains.Another possibility is that the treatment of the pigs with erythromycinmakes the pigs more sensitive for S. suis infections and a thirdpossibility is that compared to the pigs previously used, the pigs usedfor the current experiments were more sensitive for S. suis infections.

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TABLE 1 Bacterial strains and plasmids strain/plasmid relevantcharacteristics* source/reference Strain E. coli XL2 blue Stratagene S.suis 10 pathogenic serotype 2 strain Vecht et al. S735 weak-pathogenicserotype 2 reference strain Vecht et al. 24 weak-pathogenic serotype 2strain Vecht et al. Plasmid pKUN19 replication functions pUC, Amp^(R)Konings et al. pE194 Em^(R) Horinouchi et al. pMR11 pKUN19 containing S.suis mrp gene Smith et al. pCOM1 replication functions pWVO1, Em^(R)this work pCOM-L pCOM1 containing random sequences of S. suis strain 10this work pCOM-V10 pCOM1 containing S. suis strain 10 fragment selectedin pigs this work pCOM-V735 pCOM1 containing a 3.1 kb PstI-HindIIIfragment from this work S. suis strain S735 (homologous to V10)*Spc^(R): spectinomycin resistant Amp^(R): ampicillin resistant Em^(R):erythromycin resistant

TABLE 2 Virulence of S. suis library and strains in germfree pigsclinical index no. of pigs mean no. of the group in which S. suis No. ofmortality^(a) of days morbidity^(b) specific^(c) non-specific^(d) feverleukocyte was isolated from strains pigs dose (%) till death (%)symptoms symptoms index^(e) index^(f) CNS serosa joints A S735(pCOM-L) 410⁷ 100 4 100 69 91 25 n.a. 3 2 3 S735(pCOM-L) 4 10⁸ 75 7 100 50 69 2017 2 1 2 B S735(pCOM-V10) 5 10⁶ 100 1 100 100 100 54  4 5 5 5S735(pCOM1) 4 10⁶ 25 12 25 2 11 6 80 1 1 2 C S735(pCOM-V10) 5 10⁶ 100 1100 100 100 60 n.a. 5 5 5 S735(pCOM-V735) 5 10⁶ 20 15 100 40 26 17 52 11 1 S735(pCOM1) 5 10⁶ 20 16 60 11 9 11 20 1 0 0 D 24(pCOM-V10) 5 10⁶ 1002 100 50 66 42 29 3 3 5 24(pCOM-V735) 4 10⁶ 25 15 100 40 30 17 18 1 0 024(pCOM1) 5 10⁶ 20 15 20 2 14 6 21 1 0 0 E S735(pCOM-V10) 4 10⁶ 100 1100 100 100 57 n.d. 4 4 4 S735(pCOM-V10-ORF2) 4 10⁶ 100 1 100 100 84 50n.d. 4 4 4 S735(pCOM-V10-ORF3) 4 10⁶ 0 11 0 6 4 3 n.d. 0 0 0 S735(pCOM1)4 10⁶ 0 11 0 0 9 5 n.d. 0 0 0 ^(a)Percentage of pigs that died due toinfection or had to be killed for animal welfare reasons ^(b)Percentageof pigs with specific symptoms ^(c)Percentage of observations for theexperimental group in which specific symptoms (ataxia, lameness of atleast one joint and/or stillness) were observed ^(d)Percentage ofobservations for the experimental group in which non-specific symptoms(inappetite and/or depression) were observed ^(e)Percentage ofobservations for the experimental group of a body temperature of >40° C.^(f)Percentage of blood samples for the experimental group in which theconcentration of granulocytes was >10¹⁰/liter n.a.: not applicable n.d.:not determined

1. An isolated or recombinant nucleic acid comprising a sequenceselected from the group consisting of SEQ ID NO:14, primers and probesthereof and a conservatively substituted variant of SEQ ID NO: 14,wherein the variant encodes an amino acid sequence selected from thegroup consisting of SEQ ID NO:10 and SEQ ID NO:12, and wherein saidprimers and probes thereof are identifiable by hybridization at 65° C.to the sequence of SEQ ID NO:14, and washing twice with a solution of 40mM sodium phosphate (pH 7.2), 1 mM EDTA and 5% sodium dodecyl sulphatefor 30 minutes at 65° C. and washing twice with a solution of 40 mMsodium phosphate (pH 7.2), 1 mM EDTA and 1% sodium dodecyl sulphate for30 minutes at 65° C.
 2. A vector comprising the isolated or recombinantnucleic acid of claim
 1. 3. A host cell comprising the isolated orrecombinant nucleic acid of claim
 1. 4. The host cell of claim 3,wherein the host cell is of a Streptococcus origin.
 5. A host cellcomprising the vector of claim
 2. 6. An isolated or recombinant nucleicacid comprising a sequence selected from the group consisting of SEQ IDNO:14, primers and probes thereof, wherein said primers and probesthereof are identifiable by hybridization at 65° C. to the sequence ofSEQ ID NO:14, and washing twice with a solution of 40 mM sodiumphosphate (pH 7.2), 1 mM EDTA and 5% sodium dodecyl sulphate for 30minutes at 65° C. and washing twice with a solution of 40 mM sodiumphosphate (pH 7.2), 1 mM EDTA and 1% sodium dodecyl sulphate for 30minutes at 65° C.